The present invention relates to a method of inhibiting Interleukin (xe2x80x9cILxe2x80x9d)-12 signaling in inflammatory disorders, more particularly in inhibiting IL-12 signaling in CD4+ Th1 cell-mediated inflammatory disorders.
Inflammatory responses are a component of the pathogenesis of many vertebrate disorders, including those in man. In its broadest meaning, the term xe2x80x9cinflammationxe2x80x9d denotes local as well as systemic responses. Increased blood flow, vasodilation, fluid transudation from the vessels, infiltration of the tissues by leukocytes and, in some severe cases, intravascular thrombosis, damage to the blood vessels and extravasation of blood characterize local inflammation. The systemic inflammatory response, also denoted as an acute phase response, is characterized by various reactions including, for example, fever, leukocytosis and release of acute phase reactants into the serum. In severe cases, shock and death may occur. Heremans et al., Lymphokine Research 8(3): 329-333 (1989).
The inflammatory response is controlled by a variety of cellular events. Cytokines are primary factors in the cascade of events that regulate inflammatory responses. Some cytokines induce or release other known mediators of inflammation. These systems are controlled by related feed-back mechanisms. Thus, inflammatory responses are not due to a single cytokine being released in large quantities, but rather to a set of cytokines acting collectively via a network of intercellular signals to incite the inflammatory response. Ibid.
One particular cytokine, IL-12, also known as natural killer cell stimulatory factor (xe2x80x9cNKSFxe2x80x9d) and cytotoxic lymphocyte maturation factor (xe2x80x9cCLMFxe2x80x9d), is a potent immunoregulatory molecule which plays a role in a wide range of diseases. In particular, IL-12 is known to play a specific role in diseases exhibiting an inflammatory component, namely, diseases that exhibit cell-mediated inflammatory responses, such as, multiple sclerosis, diabetes, and chronic inflammatory bowel disease. Ibid.
IL-12 is produced by phagocytic cells, such as monocytes/ macrophages, B-cells and other antigen-presenting cells (xe2x80x9cAPCxe2x80x9d). Functionally, IL-12 affects both natural killer (xe2x80x9cNKxe2x80x9d) and T-lymphocytes (xe2x80x9cT cellsxe2x80x9d), and stimulates IFN-xcex3 production by both of these cell types. For example, in NK cells, IL-12 stimulates: NK cell proliferation, membrane surface antigen up-regulation, LAK cell generation and NK cell activity elevation; induces IFN-xcex3 and TNF-xcex1 production and the growth and expansion of either resting or IL-2 activated NK cells; and increases soluble p55 and soluble p75 TNF receptor production and NK cell cytotoxicity. RandD Systems Catalog, pp. 67-69 (1995).
T-cell recognition of antigen occurs via interaction of a heterodimeric (alpha/beta, or gamma/delta) receptor with short peptide antigenic determinants that are associated with major histocompatibility complex (xe2x80x9cMHCxe2x80x9d) molecules. T cells can be divided broadly into two functional categories by the presence of two mutually exclusive antigens on their cell surface, CD4 (helper) and CD8 (cytotoxic). The CD4 and CD8 antigens regulate T-cell interaction with MHC and their mutually exclusive expression derives from their strict specificity for MHC. Class II MHC-restricted T cells are primarily CD4+ and class I MHC-restricted T-cells are CD8+. The T cells further differentiate into helper, cytotoxic and suppressor cells. As mentioned above, IL-12 also affects T-cells. As mentioned above, IL-12 also affects T cells, including stimulation of T cell IFN-xcex3 production in response to antigen. T cells are broadly divided into two major subsets based on the expression of two mutually exclusive surface antigens, namely CD4 and CD8. While CD8+ T cells are associated with cytotoxicity functions, CD4+ T cells are associated with helper function and secrete various cytokines which regulate and modulate immune responses. CD4+ T cells can be further subdivided into T helper 1 (Th1) and T helper 2 (Th2) subsets, according to the profile of cytokines they secrete. Therefore, Th1 cells produce predominantly inflammatory cytokines, including IL-2, TNF-xcex1 and IFN-xcex3, while Th2 cells produce anti-inflammatory cytokines such as IL-4, IL-5, IL-10, and IL-13 which are linked to B cell growth and differentiation.
The Th1 and Th2 CD4+ T cell subsets are derived from a common progenitor cell, termed Th0 cells. During initial encounter with antigen, the differentiation into Th1 and Th2 is controlled by the opposing actions of two key cytokines, namely IL-12 and IL-4, which induce the differentiation of Th0 into Th1 and Th2, respectively. The development of Th1 and Th2 cells is primarily influenced by the cytokine milieu during the initial phase of the immune response, in which IL-12 and IL-4, respectively, play decisive roles. The cytokines produced by each Th-cell phenotype are inhibitory for the opposing phenotype. For example, Th1 cytokines enhance cell-mediated immunities and inhibit humoral immunity. Th2 cytokines enhance humoral immunity and inhibit cell-mediated immunities. Trembleau et. al., Immunology Today 16(8): 383-386 (1995).
Furthermore, CD4+ Th1 cells play a role in the pathogenesis of immunological disorders. These cells primarily secrete cytokines associated with inflammation such as IFN-xcex3, TNF-xcex1, TNF-xcex2, and IL-2. IFN-xcex3 is an important component of the inflammatory response and resultant pathology of those diseases exhibiting an inflammatory response. Heremans, et al. In addition to its role in inflammatory response, IFN-xcex3 also contributes to phagocytic cell activation (i.e., macrophage activation), and up-regulation of MHC expression on the surface of antigen-presenting cells (xe2x80x9cAPCxe2x80x9d) and other cells. Furthermore, this cytokine is implicated generally in inflammatory immune responses, and in autoimmune diseases, such as MS, specifically. Owens et al., Neurologic Clinics, Volume 13(1):51-73 (1995). Furthermore, steroid treatment broadly attenuates cytokine production, but it cannot modulate it selectively, e.g., just the Th0, the Th1 or the Th2 pathways.
IL-12 plays a role in the induction of Th1-cell-mediated autoimmunity. Recent evidence points to a critical role for IL-12 in the pathogenesis of rodent models of Th1-mediated autoimmune diseases such as type-1 diabetes, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and acute graft-versus-host disease. Thus, Th1 cells are considered to be involved in the induction of experimental autoimmune diseases. Evidence for this is based on adoptive transfer experiments demonstrating the CD4+ cells producing Th1-type lymphokines can transfer disease, as shown in models of experimental autoimmune disease, such as experimental allergic encephalomyelitis (xe2x80x9cEAExe2x80x9d) (also known as experimental allergic encephalitis) and insulin-dependent diabetes mellitus (xe2x80x9cIDDMxe2x80x9d). Trinchieri, Annu. Rev. Immunol. 13(1):251-276 (1995). EAE is an inflammatory T cell mediated, paralytic, demyelinating, autoimmune disease that can be induced in a number of rodents as well as primates. Owens et al. One of the ways that EAE can be induced is by immunization of animals with myelin basic protein (xe2x80x9cMBPxe2x80x9d). Ibid. Administration of IL-12 induces rapid onset of IDDM in 100% of NOD female mice. Trinchieri. Thus, one goal of immunotherapy has been to limit the inflammatory response while leaving the specificity of the immune system, deemed necessary for host protection, intact.
For example, steroid therapy is the most common treatment for one such IL-12 mediated disease, Multiple Sclerosis (xe2x80x9cMSxe2x80x9d), particularly, corticosteroids. This suggests that steroids alter the trafficking of cells into the brain or reduce the secretion of cytokines by inflammatory cells in areas of inflammation. Ibid. Although their effect in reversing some of the acute symptoms of autoimmune disease, such as MS, are well known, their side effects have precluded long-term use. Ibid.
Other treatments that target immune system components include lymphocyte cytotoxic drugs such as cyclophosphamide and azathioprine. These drugs act as sledgehammers, suppressing the entire immune system, with the problems that attend broad-spectrum immunosuppression. The same problems also are likely with newer therapies such as cyclosporine, anti-CD4 monoclonal antibodies, and others. Other treatments for IL-12 mediated diseases, including MS, can involve the administration of anti-IL-12 antagonists such as antibodies. Anti-IL-12 antibodies have been shown to inhibit the development of IDDM and EAE. Trinichieri. However, antibody based immunotherapy may result in immune complex formation and deposition, thus leading to glomerulonephritis, vasculitis and arthritis.
Still other treatments for IL-12 mediated diseases involve the phosphodiesterase inhibitor, pentoxyifylline (xe2x80x9cPTXxe2x80x9d). For example, PTX has been used as an anti-inflammatory agent in T-cell mediated autoimmune disease, exemplified by the animal model of EAE in Lewis rats. Rott et al., Eur. J. Immunol. 23:1745-1751, (1993). EAE in rodents is a well-characterized animal model for the study of demyelinating inflammation in the central nervous system (xe2x80x9cCNSxe2x80x9d) and autoimmune diseases in general, including MS. Ibid. PTX is able to suppress efficiently the activation of Th1 cells in vitro and in vivo without resulting in an overall immunosuppression or causing obvious severe side effects. Ibid. Pretreatment with IFN-xcex3 reduced the suppressive effects of pentoxifylline on production of IL-12, and thus, is not a suitable therapy when there occurs an ongoing production of IFN-xcex3. Moller et al., Immunology 91:197-203 (1997).
Thus, there remains a need for compounds and methods that singularly inhibit the deleterious effects of inflammatory responses mediated by specific cytokines, such as IL-12, without effecting the other components of the immune system deemed necessary for protection of the host. The present invention fulfills this need and provides for further related advantages.
It is an object of the present invention to provide a method of inhibiting IL-12 signaling in a mammal having a CD4+ Th1 cell-mediated inflammatory response by administering a signal inhibiting amount of the compound of the following formula: 
wherein, R1 is H, CH3, sulfate, phosphate, or salt thereof; R2 is alkyl (C1-12), alkoxyalkyl (C1-11), dialkoxyalkyl, CH2C6H5, xe2x80x94CH2-furan, biotin; and R3 is H, CH3 or CH2C6H5. In accomplishing this and other objectives, the present invention provides a method for affecting The inflammatory response associated with CD4+ Th1 cell-mediated diseases; without affecting The other components of the immune system, deemed necessary for host protection. The present methods short-circuit the inflammatory cascade by inhibiting IL-12-dependent Th1 development, emphasizing the inventive methods importance in disease therapy by inhibiting IL-12 signaling in the regulation of CD4+ Th1-mediated inflammatory disorders.